title: Increased yield of high-purity and active tetrameric recombinant human EC-SOD by solid phase refolding.
abstract: Superoxide dismutase (SOD) removes damaging reactive oxygen species from the cellular environment by catalyzing the dismutation of two superoxide radicals to hydrogen peroxide and oxygen. Extracellular superoxide dismutase (EC-SOD) is a tetramer and is present in the extracellular space and to a lesser extent in the extracellular fluids. Increasing therapeutic applications for recombinant human extracellular superoxide dismutase (rEC-SOD) has broadened interest in optimizing methods for its purification, with a native conformation of tetramer. We describe a solid phase refolding procedure that combines immobilized metal affinity chromatography (IMAC) and gel filtration chromatography in the purification of rEC-SOD from Escherichia coli. The purified rEC-SOD tetramer from the Ni(2+)-column chromatography is refolded in Tris buffer. This method yields greater than 90% of the tetramer form. Greater than 99% purity is achieved with further purification over a Superose 12PC 3.2/30 column to obtain the tetramer and specific activities as determined via DCFHDA assay. The improved yield of rEC-SOD in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.
authors.affiliation: Laboratory of Dermato-Immunology, College of Medicine, The Catholic University of Korea, Seoul 130-701, Korea.
authors.forename: Kang
authors.initials: K
authors.lastname: Ryu
authors.forename: Young-Hoon
authors.initials: YH
authors.lastname: Kim
authors.forename: Younghwa
authors.initials: Y
authors.lastname: Kim
authors.forename: Joon-Seok
authors.initials: JS
authors.lastname: Lee
authors.forename: Byeongwook
authors.initials: B
authors.lastname: Jeon
authors.forename: Tae-Yoon
authors.initials: TY
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D013482:Superoxide Dismutase